Breathing is a robust and adaptive behavior, and its complexity is often overlooked because of its constant presence. Recent work suggests that breathing is produced by two distinct rhythmic centers in the brainstem. One hypothesis is that each center is responsible for a specific aspect of breathing: one center is responsible for inspiration (preBotzinger complex, preBotC), the other is responsible for active expiration (parafacial respiratory group, pFRG). To date, the connectivity between these two centers is unclear as is the precise role of the pFRG. To begin to understand the interplay between the preBotC and the pFRG, we will characterize the direct and indirect anatomical connectivity between the two centers by injecting dyes of different colors into the different centers and performing tract-tracing experiments (AimlA). We will obtain electrical recordings from single neurons in each center and reconstruct their morphology (AimlB). We will thus gain a clearer picture of how the two centers are connected, including the identification of indirect connections in the medulla. For individual neurons in each center we will begin to link firing patterns to specific anatomical projections. Both sets of experiments will increase our understanding of the organization of the neural circuit for breathing. To determine the role of each rhythmic center in breathing, we will use drugs to selectively and reversibly speed up or slow down one rhythmic center (pFRG or preBotC) while continuously monitoring breathing-related motor activity on distinct spinal nerves (Aim2A). Using drugs, we will reversibly silence the pFRG and characterize changes in the breathing rhythm and pattern. Related to these experiments, we will provide further quantification of the breathing rhythm and pattern in transgenic mice (Krox20-/-) that have pFRG/RTN pathology (Aim2B). If our running hypothesis is correct, the mice without a functional pFRG should have breathing rhythms that are similar to those observed in rats where the pFRG is reversibly silenced. Last, we will optically monitor the breathing centers when one neuron is stimulated to look at within center and between center connectivity (Aim3). Public health relevance: ln humans, breathing is essential to life and requires that the nervous system generate a reliable and robust rhythm. The proposed study aims to understand how two rhythmic brainstem centers are connected and how they interact to produce breathing. This information can aid in the development of rational therapeutics for central sleep disorders and will improve our understanding of the neural systems that are involved in the breathing rhythm.